Impact of Joule Heating on Electrokinetic Lateral Flow Assay

Lateral flow assays (LFAs) are indispensable tools for point-of-care testing. However, their typically high limit of detection (LOD) restricts their applicability in many applications. Recent advances have shown that coupling LFAs with electrophoresis can lower the LOD by orders of magnitude without compromising the assay's simplicity, speed, or cost. Nevertheless, Joule heating resulting from the applied electric current unavoidably raises the temperature of the test strip, which may lead to biomolecule denaturation and a deterioration in sensitivity. We used a two-stage, double-antigen lateral flow assay for the detection of IgG antibodies against hepatitis B surface antigen (HBsAg) in human serum. In the first stage, IgG antibodies reacted with immobilized HBsAg during capillary flow. In the second stage, protein G conjugated with Au nanoparticles was electrophoretically driven through the test strip, resulting in the formation of labeled immune complexes. The second stage was accompanied by Joule heating of the membrane. We demonstrate that membrane overheating (exceeding 80 °C) causes a 42-fold increase in the LOD (decrease of sensitivity), along with the emergence of false-positive results. In this study, we identify the key parameters influencing heating, such as buffer composition and ionic strength, common additives (e.g., surfactants, electroosmotic flow mediators, cations), applied voltage, and test strip geometry. These findings offer practical guidance for the development of electrokinetic assays, enabling operation within a controlled thermal regime and eliminating the need for extensive thermal profiling.